Chronic fatigue syndrome (CFS) is a complex multisymptom disorder that lacks objective blood-based biomarkers to use in diagnosis or clinical management of symptoms. Until recently, the peripheral processes involved in sensing muscle fatigue and pain were unknown; however, animal model studies by our group and others have clarified that a complex of ion channel receptors (including Acid Sensing Ion Channel-3 or ASIC3, ATP-sensing Purinergic 2X or P2X, and TRPV1 or capsaicin receptors) working together can detect increased metabolic by-products of work in muscle (ATP, lactate and pH changes). The numbers of these ion channel receptors are not fixed but can increase markedly in response to inflammatory processes or exercise. Supported by an R21 grant, we completed the first translational study to show dysregulation of these receptors in CFS after 25 min of moderate exercise (at 70% of age-predicted maximum heart rate). Using blood samples from 0.5 through 48 hours after exercise, CFS patients but not healthy controls showed both rapid and sustained increases in expression of these fatigue-sensing ion channel receptors present on leukocytes, together with increases in adrenergic alpha-2a, beta-1 and beta-2 receptors, and of both pro- and anti-inflammatory cytokines (Light, et al., 2009; White et al, 2010). We also recently showed that this post-exercise gene expression profile clearly differentiated patients with CFS from those with multiple sclerosis or fibromyalgia without comorbid CFS. The present study will add 140 additional subjects (including 70 CFS patients) to our existing database of 178 subjects that already includes 50 CFS patients and 50 controls, all studied using the same moderate exercise challenge, with blood sampling before and at 4 times after exercise. We plan to use both full genomic microarrays plus qPCR with targeted genes including ion channel and adrenergic receptors, immune genes, plus several new genes including XPR1, neuropeptide Y, HPA axis receptors, and transcription factors. Our aims are to examine whether: 1) using stringent STARD criteria for biomarker evaluation, CFS patients can be clearly differentiated from healthy controls using our post-exercise gene expression profiles, 2) CFS patients also differ from patients with major depressive disorder and patients with prostate cancer who have cancer-related fatigue; 3) these profiles reliably differentiate women vs. men with CFS, and subgroups of CFS patients identified by post-exercise increases vs. decreases in alpha-2a adrenergic receptor expression. This investigation will provide a strong test of these gene expression measures as diagnostic biomarkers in CFS as a whole and in key subgroups. It will also lay a foundation for further translational research on dysregulated pathways that may initiate, maintain or worsen symptoms of CFS, and provide potential targets for effective therapeutic intervention.